Engineering CD4+ T cells to develop a novel immunotherapy for pancreatic cancer

Project Summary Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest forms of cancer, with a dismal 5-year survival rate of only 13%. Despite advances in cancer immunotherapy, the efficacy of adoptive cellular therapies (ACT), including chimeric antigen receptor (CAR) T cell therapy, in PDAC is limited due to the dense desmoplastic stroma and immunosuppressive tumor microenvironment (TME). This project aims to develop and explore novel approaches to enhance the effectiveness of ACT for PDAC. The F99 phase (Aim 1) focuses on assessing the efficacy of mesothelin-directed CD26+CD4+ CAR T cells in murine PDAC models. CD26+CD4+ T cells have shown favorable stem-memory phenotypes and polyfunctional cytokine secretion capabilities. We hypothesize that CD26 on CAR T cells plays an active role in reshaping the TME through its enzymatic activity, which functionally alters immunosuppressive peptides. Our approach involves using congenic markers to distinguish between donor and host cells, allowing for detailed immune profiling via flow cytometry. We will assess tumor growth over time using luciferase-expressing PDAC cells and bioluminescence imaging. To elucidate the importance of CD26 enzymatic activity, we will employ systemic inhibition using sitagliptin and evaluate its impact on CAR T cell efficacy, persistence, and memory phenotypes. Additionally, we will investigate the potential synergistic effects of co-infusing CD26+CD4+ CAR T cells with CD8+ T cells to determine whether multifaceted T cell-mediated interactions enhance cellular therapy efficacy. The K00 phase (Aim 2) builds upon my predoctoral work in ACT development to characterize the impact of the microbiome on ACT outcomes in PDAC. Recent evidence suggests that the gut microbiome significantly influences the efficacy of cancer immunotherapies; I therefore seek to identify specific microbial signatures associated with improved responses to ACT. I will employ 16S rRNA sequencing and metagenomic analysis to characterize the gut microbiome composition in PDAC mouse models undergoing different adoptive cell therapies. I will also investigate the efficacy of ACT in response to different microbiomes, manipulated with antibiotics or by addition of defined microbial consortia into germ-free mice. Mechanisms by which the microbiome influences immune cell function will be interrogated both in vitro and in vivo. Based on these findings, I aim to develop novel microbiome- modulating strategies, such as targeted prebiotics or probiotics or novel engineered ACT strategies, to enhance the efficacy of adoptive cell therapies in PDAC. This research has the potential to significantly advance our understanding of adoptive cell therapies for PDAC and other solid tumors. By unraveling the intricacies of CD26- mediated immunomodulation (F99) and exploring the role of the microbiome in ACT treatment outcomes (K00), we aim to develop more effective strategies for overcoming the challenges posed by the PDAC tumor microenvironment. Ultimately, this work could lead to the development of novel, more potent adoptive cell therapies in pancreatic cancer, offering new hope for patients facing this devastating disease.